干扰素基因刺激因子通过肺巨噬细胞胞葬功能调控急性肺损伤小鼠修复的研究

doi:10.3877/cma.j.issn.1674-6880.2024.02.002

目的

本研究旨在探讨干扰素基因刺激因子（STING）对小鼠急性肺损伤修复过程的作用，并深入研究其对小鼠巨噬细胞胞葬功能的影响。

方法

选取25只10 ~ 12周龄的C57BL/6小鼠，并随机分为D0组、D1组、D3组、D5组、D7组，每组5只，分别给予气管内注射0.5 mg/kg脂多糖（LPS）0、1、3、5、7 d后收集小鼠样本。另外，分别选择野生型STING^+/+和STING基因缺失型（STING^-/-）小鼠各5只，分为STING^+/+ + LPS组和STING^-/- + LPS组，同样给予气管内注射0.5 mg/kg LPS诱导5 d后收取小鼠样本。通过苏木素-伊红（HE）染色观察小鼠肺组织的损伤病理变化，并检测小鼠肺泡灌洗液细胞数、中性粒细胞数、蛋白浓度来评估肺损伤程度；经小鼠气管注射凋亡的中性粒细胞，并于3 h后收集肺泡灌洗液细胞进行流式细胞术检测，观察巨噬细胞的胞葬功能；采用免疫荧光染色法对肺组织进行TUNEL和F4/80双染评估胞葬作用；并采用巴氏染色法观察肺泡巨噬细胞的胞葬现象。

结果

C57BL/6小鼠诱导急性肺损伤后3 d达到肺损伤的顶峰，5 d后开始修复，并在不治疗的情况下，于7 d内修复基本完成。小鼠急性肺损伤5 d后，与STING^+/+ + LPS组小鼠相比，STING^-/- + LPS组小鼠的肺损伤评分、肺泡灌洗液总蛋白浓度、肺泡灌洗液总细胞数、肺泡灌洗液中性粒细胞数均较STING^+/+ + LPS组更低（t = 3.257、2.926、3.946、2.669，P = 0.012、0.019、0.004、0.028）。流式细胞术及免疫荧光染色检测结果显示，STING^-/- + LPS组小鼠肺泡巨噬细胞胞葬率均显著高于STING^+/+ + LPS组（t = 3.143、6.963，P = 0.016、< 0.001）。

结论

STING缺失可能通过促进巨噬细胞胞葬功能，从而调控急性肺损伤的修复过程。本研究有望为深入理解STING在肺部损伤和修复中的作用提供新的见解。

关键词: 急性肺损伤, 干扰素基因刺激因子, 巨噬细胞, 胞葬, 小鼠

Objective

To investigate the impact of the stimulator of interferon genes (STING) on the repair process of acute lung injury in mice and its influence on macrophages efferocytosis.

Methods

Twenty-five C57BL/6 mice aged 10-12 weeks were randomly divided into a D0 group, a D1 group, a D3 group, a D5 group and a D7 group, with five mice in each group, and the mice samples were collected after 0, 1, 3, 5 and 7 days of an intratracheal injection of 0.5 mg/kg lipopolysaccharide (LPS) respectively. In addition, five STING wild-type (STING^+/+) mice and five STING knockout (STING^-/-) mice were selected and divided into a STING^+/+ + LPS group and a STING^-/- + LPS group, and the mice samples were collected after 5 days of an intratracheal injection of 0.5 mg/kg LPS. Hematoxylin eosin (HE) staining was employed to observe the pathological changes in lung tissue, and the cell count, neutrophil count and protein concentration in alveolar lavage fluid were detected to access the degree of lung injury. Apoptotic neutrophils were injected into the trachea of mice, and alveolar lavage fluid cells were collected 3 hours later for flow cytometry to determine the efferocytosis rate of macrophages. TUNEL and F4/80 double staining of lung tissues were conducted via immunofluorescence to evaluate efferocytosis. The phenomenon of macrophage efferocytosis was observed through Pap staining.

Results

The C57BL/6 mice reached the peak of lung injury 3 days after induction of acute lung injury and began to repair after 5 days, and in the absence of treatment, the repair was basically completed within 7 days. After 5 days of acute lung injury, the lung tissue injury score and the protein concentration, total cell count and neutrophil count in alveolar lavage fluid of mice in the STING^-/- + LPS group were lower than those in the STING^+/+ + LPS group (t = 3.257, 2.926, 3.946, 2.669; P = 0.012, 0.019, 0.004, 0.028). Flow cytometry and immunofluorescence staining showed that the efferocytosis rates of alveolar macrophages in the STING^-/- + LPS group were significantly higher than those in the STING^+/+ + LPS group (t = 3.143, 6.963; P = 0.016, < 0.001).

Conclusions

Deletion of STING may regulate the repair process of acute lung injury through enhancing macrophage efferocytosis. Therefore, this study provides new insights into the role of STING in the lung injury and repair.

Key words: Acute lung injury, Stimulator of interferon genes, Macrophage, Efferocytosis, Mice

图1 各组小鼠肺损伤后的病理变化（HE染色　× 200）注：HE.苏木素-伊红；LPS.脂多糖；a ~ e图分别为D0组、D1组、D3组、D5组和D7组小鼠肺损伤病理结果；D0组、D1组、D3组、D5组和D7组分别为LPS诱导0、1、3、5、7 d；a图显示D0组小鼠肺泡结构正常；b图显示D1组小鼠肺泡壁增厚，较多炎症细胞浸润；c图显示D3组小鼠肺泡壁明显增厚，形态结构破坏，大量炎症细胞浸润；d图显示D5组小鼠肺泡壁稍破坏，少量炎症细胞浸润；e图显示D7组小鼠肺组织结构趋于正常

图2 两组小鼠肺部炎症的变化（n = 5）注：STING.干扰素基因刺激因子；LPS.脂多糖；HE.苏木素-伊红；BALF.肺泡灌洗液；a、b图分别为STING^+/+ + LPS组和STING^-/- + LPS组小鼠肺组织病理变化（HE染色　× 200）；a图显示STING^+/+ + LPS组小鼠肺泡结构明显受损，肺泡壁明显增厚，较多炎症细胞浸润；b图显示STING^-/- + LPS组小鼠肺泡结构受损程度较轻，肺泡壁一般增厚，可见少量炎症细胞浸润；c图为两组小鼠肺损伤评分比较；d图为两组小鼠BALF总蛋白浓度比较；e图为两组小鼠BALF总细胞数比较；f图为两组小鼠BALF中性粒细胞数比较；与STING^+/+ + LPS组比较，^aP < 0.05

图3 STING^+/+ + LPS组和STING^-/- + LPS组小鼠肺泡灌洗液细胞病理结果（巴氏染色　× 400）注：STING.干扰素基因刺激因子；LPS.脂多糖；a图显示STING^+/+ + LPS组小鼠有较少的肺泡巨噬细胞吞噬中性粒细胞现象（红色箭头指示）；b图显示STING^-/- + LPS组小鼠肺泡巨噬细胞吞噬中性粒细胞现象明显增多（红色箭头指示）

图4 流式细胞术检测两组小鼠肺泡巨噬细胞胞葬率（n = 5）注：STING.干扰素基因刺激因子；LPS.脂多糖；a、b图分别为STING^+/+ + LPS组和STING^-/- + LPS组小鼠流式细胞检测图；c图为两组小鼠肺泡巨噬细胞胞葬率比较；与STING^+/+ + LPS组比较，^aP < 0.05

图5 两组小鼠肺组织巨噬细胞胞葬情况（n = 5）注：STING.干扰素基因刺激因子；LPS.脂多糖；a、b图分别为STING^+/+ + LPS组和STING^-/- + LPS组小鼠肺组织免疫荧光染色结果[F4/80标记巨噬细胞（红色荧光），TUNEL标记凋亡的中性粒细胞（绿色荧光），× 400]；a图显示STING^+/+ + LPS组小鼠F4/80与TUNEL双染细胞较少（白色箭头指示）；b图显示STING^-/- + LPS组F4/80与TUNEL双染细胞明显增多（白色箭头指示）；c图为两组小鼠肺组织巨噬细胞胞葬率比较；与STING^+/+ + LPS组比较，^aP < 0.05

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Stimulator of interferon genes affects acute lung injury repair in mice through pulmonary macrophage efferocytosis

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Stimulator of interferon genes affects acute lung injury repair in mice through pulmonary macrophage efferocytosis